Label-free nonlinear microscopy offers a powerful tool for the biomedical sciences. It enables investigations of cells and tissues using signals that emerge from endogenous biomolecules and microstructures to derive contrast, thereby preserving the physiological viability and functionality of specimens. Today, the most advanced label-free nonlinear microscopes are multimodal imaging platforms that capitalize on the heterogeneity of biological specimens, capturing not one but many nonlinear signals. Thus, label-free multimodal nonlinear imaging attains a contrast palette with complementary signals, delivering data-rich images that not only allow spatial unmixing and quantification of biochemical species but also unleash the power of correlation analyses and artificial intelligence to extract further information from specimens. In this Perspective, we recap the nonlinear contrast palette and compare the two technological strategies often used to acquire multimodal nonlinear images: a sequential approach vs a simultaneous approach. We then present their strengths and weaknesses and discuss emerging computational strategies that enhance the interpretability of multimodal data.

Increasing the energy efficiency and reducing the footprint of on-chip photodetectors enable dense optical interconnects for emerging computational and sensing applications. While heterojunction phototransistors (HPTs) exhibit high energy efficiency and negligible excess noise factor, their gain-bandwidth product (GBP) has been inferior to that of avalanche photodiodes at low optical powers. Here, we demonstrate that utilizing type-II energy band alignment in an Sb-based HPT results in six times smaller junction capacitance per unit area and a significantly higher GBP at low optical powers. These type-II HPTs were scaled down to 2 m in diameter and fully integrated with photonic waveguides on silicon. Thanks to their extremely low dark current and high internal gain, these devices exhibit a GBP similar to the best avalanche devices (∼270 GHz) but with one order of magnitude better energy efficiency. Their energy consumption is about 5 fJ/bit at 3.2 Gbps, with an error rate below 10 at -25 dBm optical power at 1550 nm. These features suggest new opportunities for creating highly efficient and compact optical receivers based on phototransistors with type-II band alignment.

Mid-infrared microscopy is an important tool for biological analyses, allowing a direct probe of molecular bonds in their low energy landscape. In addition to the label-free extraction of spectroscopic information, the application of broadband sources can provide a third dimension of chemical specificity. However, to enable widespread deployment, mid-infrared microscopy platforms need to be compact and robust while offering high speed, broad bandwidth, and high signal-to-noise ratio. In this study, we experimentally showcase the integration of a broadband, high-repetition-rate dual-comb spectrometer (DCS) in the mid-infrared range with a scanning microscope. We employ a set of 1-GHz mid-infrared frequency combs, demonstrating their capability for high-speed and broadband hyperspectral imaging of polymers and ovarian tissue. The system covers at with 12.86 kHz spectra acquisition rate and spatial resolution. Taken together, our experiments and analysis elucidate the trade-off between bandwidth and speed in DCS as it relates to microscopy. This provides a roadmap for the future advancement and application of high-repetition-rate DCS hyperspectral imaging.

Brillouin spectroscopy has become an important tool for mapping the mechanical properties of biological samples. Recently, stimulated Brillouin scattering () measurements have emerged in this field as a promising technology for lower noise and higher speed measurements. However, further improvements are fundamentally limited by constraints on the optical power level that can be used in biological samples, which effectively caps the gain and signal-to-noise ratio () of biological measurements. This limitation is compounded by practical limits on the optical probe power due to detector saturation thresholds. As a result, -based measurements in biological samples have provided minimal improvements (in noise and imaging speed) compared with spontaneous Brillouin microscopy, despite the potential advantages of the nonlinear scattering process. Here, we consider how a spectrometer can circumvent this fundamental trade-off in the low-gain regime by leveraging the polarization dependence of the interaction to effectively filter the signal from the background light via the polarization pulling effect. We present an analytic model of the polarization pulling detection scheme and describe the trade-space unique to Brillouin microscopy applications. We show that an optimized receiver design could provide >25× improvement in compared to a standard receiver in most typical experimental conditions. We then experimentally validate this model using optical fiber as a simplified test bed. With our experimental parameters, we find that the polarization pulling scheme provides 100× higher than a standard receiver, enabling 100× faster measurements in the low-gain regime. Finally, we discuss the potential for this proposed spectrometer design to benefit low-gain spectroscopy applications such as Brillouin microscopy by enabling pixel dwell times as short as 10 s.

Chlorine exposure can cause severe airway injuries. While the acute effects of chlorine inhalation are well-documented, the structural changes resulting from the post-acute, high-level chlorine exposure remain less understood. Airway sloughing is one of the standards for doctors to evaluate the lung function. Here, we report the application of a high-resolution swept-source optical coherence tomography system to investigate the progression of injury based on airway sloughing evaluation in a chlorine inhalation rabbit model. This system employs a 1.2 mm diameter flexible fiberoptic endoscopic probe via an endotracheal tube to capture large airway anatomical changes before and as early as 30 min after acute chlorine exposure. We conducted an animal study using New Zealand white rabbits exposed to acute chlorine gas (800 ppm, 6 min) during ventilation and monitored them using optical coherence tomography (OCT) for 6 h. To measure the volume of airway sloughing induced by chlorine gas, we utilized deep learning for the segmentation task on OCT images. The results showed that the volume of chlorine induced epithelial sloughing on rabbit tracheal walls initially increased, peaked around 30 min, and then decreased. Furthermore, we utilized a spectral encoded interferometric microscopy system to study airway cilia beating dynamics based on Doppler shift, aiding in elucidating how chlorine gas affects cilia beating function. Cilia movability and beating frequency were decreased because of the epithelium damage. This quantitative approach has the potential to enhance the diagnosis and monitoring of injuries from toxic gas inhalation and to evaluate the efficacy of antidote treatments for these injuries.

This tutorial reviews the rapidly growing field of optical photothermal infrared (O-PTIR) spectroscopy and chemical imaging. O-PTIR is an infrared super-resolution measurement technique where a shorter wavelength visible probe is used to measure and map infrared (IR) absorption with spatial resolution up to 30× better than conventional techniques such as Fourier transform infrared and direct IR laser imaging systems. This article reviews key limitations of conventional IR instruments, the O-PTIR technology breakthroughs, and their origins that have overcome the prior limitations. This article also discusses recent developments in expanding multi-modal O-PTIR approaches that enable complementary Raman spectroscopy and fluorescence microscopy imaging, including wide-field O-PTIR imaging with fluorescence-based detection of IR absorption. Various practical subjects are covered, including sample preparation techniques, optimal measurement configurations, use of IR tags/labels and techniques for data analysis, and visualization. Key O-PTIR applications are reviewed in many areas, including biological and biomedical sciences, environmental and microplastics research, (bio)pharmaceuticals, materials science, cultural heritage, forensics, photonics, and failure analysis.

We present deep-ultraviolet Fourier ptychography (DUV-FP) for high-resolution chemical imaging of biological specimens in their native state without exogenous stains. This approach uses a customized 265-nm DUV LED array for angle-varied illumination, leveraging the unique DUV absorption properties of biomolecules at this wavelength region. We implemented a robust feature-domain optimization framework to overcome common challenges in Fourier ptychographic reconstruction, including vignetting, pupil aberrations, stray light problems, intensity variations, and other systematic errors. By using a 0.12 numerical aperture low-resolution objective lens, our DUV-FP prototype can resolve the 345-nm linewidth on a resolution target, demonstrating at least a four-fold resolution gain compared to the captured raw images. Testing on various biospecimens demonstrates that DUV-FP significantly enhances absorption-based chemical contrast and reveals detailed structural and molecular information. To further address the limitations of conventional FP in quantitative phase imaging, we developed a spatially coded DUV-FP system. This platform enables true quantitative phase imaging of biospecimens with DUV light, overcoming the non-uniform phase response inherent in traditional microscopy techniques. The demonstrated advancements in high-resolution, label-free chemical imaging may accelerate developments in digital pathology, potentially enabling rapid, on-site analysis of biopsy samples in clinical settings.

The applications of ultrafast optics to biomedical microscopy have expanded rapidly in recent years, including interferometric techniques like optical coherence tomography and microscopy (OCT/OCM). The advances of ultra-high resolution OCT and the inclusion of OCT/OCM in multimodal systems combined with multiphoton microscopy have marked a transition from using pseudo-continuous broadband sources, such as superluminescent diodes, to ultrafast supercontinuum optical sources. We report anomalies in the dispersion profiles of low-coherence ultrafast pulses through long and non-identical arms of a Michelson interferometer that are well beyond group delay or third-order dispersions. This chromatic anomaly worsens the observed axial resolution and causes fringe artifacts in the reconstructed tomograms in OCT/OCM using traditional algorithms. We present DISpersion COmpensation Techniques for Evident Chromatic Anomalies (DISCOTECA) as a universal solution to address the problem of chromatic dispersion mismatch in interferometry, especially with ultrafast sources. First, we demonstrate the origin of these artifacts through the self-phase modulation of ultrafast pulses due to focusing elements in the beam path. Next, we present three solution paradigms for DISCOTECA: optical, optoelectronic, and computational, along with quantitative comparisons to traditional methods to highlight the improvements to the dynamic range and axial profile. We explain the piecewise reconstruction of the phase mismatch between the arms of the spectral-domain interferometer using a modified short-term Fourier transform algorithm inspired by spectroscopic OCT. Finally, we present a decision-making guide for evaluating the utility of DISCOTECA in interferometry and for the artifact-free reconstruction of OCT images using an ultrafast supercontinuum source for biomedical applications.

Optical systems use acousto-optic deflectors (AODs) mostly for fast angular scanning and spectral filtering of laser beams. However, AODs may transform laser light in much broader ways. When time-locked to the pulsing of low repetition rate laser amplifiers, AODs permit the holographic reconstruction of 1D and pseudo-two-dimensional (ps2D) intensity objects of rectangular shape by controlling the amplitude and phase of the light field at high (20-200 kHz) rates for microscopic light patterning. Using iterative Fourier transformations (IFTs), we searched for AOD-compatible holograms to reconstruct the given ps2D target patterns through either phase-only or complex light field modulation. We previously showed that phase-only holograms can adequately render grid-like patterns of diffraction-limited points with non-overlapping diffraction orders, while side lobes to the target pattern can be cured with an apodization mask. Dense target patterns, in contrast, are typically encumbered by apodization-resistant speckle noise. Here, we show the denoised rendering of dense ps2D objects by complex acousto-optic holograms deriving from simultaneous optimization of the amplitude and phase of the light field. Target patterns lacking ps2D symmetry, although not translatable into single holograms, were accessed by serial holography based on a segregation into ps2D-compatible components. The holograms retrieved under different regularizations were experimentally validated in an AOD random-access microscope. IFT regularizations characterized in this work extend the versatility of acousto-optic holography for fast dynamic light patterning.

Raman scattering has found renewed interest owing to the development of gas-filled hollow-core fibers, which constitute a unique platform for exploration of novel ultrafast nonlinear phenomena beyond conventional solid-core-fiber and free-space systems. Much progress has been made through models for particular interaction regimes, which are delineated by the relation of the excitation pulse duration to the time scales of the Raman response. However, current experimental settings are not limited to one regime, prompting the need for tools spanning multiple regimes. Here, we present a theoretical framework that accomplishes this goal. The theory allows us to review recent progress with a fresh perspective, makes new connections between distinct temporal regimes of Raman scattering, and reveals new degrees of freedom for controlling Raman physics. Specific topics that are addressed include transient Raman gain, the interplay of electronic and Raman nonlinearities in short-pulse propagation, and interactions of short pulses mediated by phonon waves. The theoretical model also accommodates vector effects, which have been largely neglected in prior works on Raman scattering in gases. The polarization dependence of transient Raman gain and vector effects on pulse interactions via phonon waves is investigated with the model. Throughout this Perspective, theoretical results are compared to the results of realistic numerical simulations. The numerical code that implements the new theory is freely available. We hope that the unified theoretical framework and numerical tool described here will accelerate the exploration of new Raman-scattering phenomena and enable new applications.

Optical diffraction tomography is a powerful technique to produce 3D volumetric images of biological samples using contrast produced by variations in the index of refraction in an unlabeled specimen. While this is typically performed with coherent illumination from a variety of angles, interest has grown in partially coherent methods due to the simplicity of the illumination and the computation-free axial sectioning provided by the coherence window of the source. However, such methods rely on the symmetry or discretization of a source to facilitate quantitative analysis and are unable to efficiently handle arbitrary illumination that may vary asymmetrically in angle and continuously in the spectrum, such as diffusely scattered or thermal sources. A general broadband theory may expand the scope of illumination methods available for quantitative analysis, as partially coherent sources are commonly available and may benefit from the effects of spatial and temporal incoherence. In this work, we investigate partially coherent tomographic phase microscopy from arbitrary sources regardless of angular distribution and spectrum by unifying the effects of spatial and temporal coherence into a single formulation. This approach further yields a method for efficient computation of the overall systems' optical transfer function, which scales with ( ), down from ( ) for existing convolutional methods, where is the number of spatial voxels in 3D space and is the number of discrete wavelengths in the illumination spectrum. This work has important implications for enabling partially coherent 3D quantitative phase microscopy and refractive index tomography in virtually any transmission or epi-illumination microscope.

Semiconductor-based laser particles (LPs) with exceptionally narrowband spectral emission have been used in biological systems for cell tagging purposes. Fabrication of these LPs typically requires highly specialized lithography and etching equipment, and is typically done in a cleanroom environment, hindering the broad adoption of this exciting new technology. Here, using only easily accessible laboratory equipment, we demonstrate a simple layer-by-layer fabrication strategy that overcomes this obstacle. We start from an indium phosphide (InP) substrate with multiple epitaxial indium gallium arsenide phosphide (InGaAsP) layers which are sequentially processed to yield LPs of various compositions and spectral properties. The LPs isolated from each layer are characterized, exhibiting excellent optical properties with lasing emission full width at half maximum as narrow as < 0.3 nm and typical thresholds of approximately 6 pJ upon excitation using a 3 ns pulse duration 1064 nm pump laser. The high quality of these particles renders them suitable for large-scale biological experiments including those requiring spectral multiplexing.

Brillouin spectroscopy has emerged as a promising modality to noninvasively probe the mechanical properties of biologically relevant materials. Stimulated Brillouin scattering (SBS) has the potential to improve measurement speed and resolution by exploiting a resonant amplification of the scattered signal, yet current SBS spectrometers have not provided significant improvements due to fundamental and practical limitations of illumination and detection parameters. To overcome this challenge, here we derive a signal localization theory for the Brillouin spectral domain and accordingly design an SBS spectrometer with much improved performances compared to state-of-the-art systems. We present experimental and simulated data validating our theory, which result in a tenfold improvement in acquisition speed, or an order of magnitude improved spectral precision, for SBS spectral measurements when properly optimizing the SBS photon detection architecture.

The field of implantable optical neural interfaces has recently enabled the interrogation of neural circuitry with both cell-type specificity and spatial resolution in sub-cortical structures of the mouse brain. This generated the need to integrate multiple optical channels within the same implantable device, motivating the requirement of multiplexing and demultiplexing techniques. In this article, we present an orthogonalization method of the far-field space to introduce mode-division demultiplexing for collecting fluorescence from the implantable tapered optical fibers. This is achieved by exploiting the correlation between the transversal wavevector of the guided light and the position of the fluorescent sources along the implant, an intrinsic property of the taper waveguide. On these bases, we define a basis of orthogonal vectors in the Fourier space, each of which is associated with a depth along the taper, to simultaneously detect and demultiplex the collected signal when the probe is implanted in fixed mouse brain tissue. Our approach complements the existing multiplexing techniques used in silicon-based photonics probes with the advantage of a significant simplification of the probe itself.

Miniature handheld imaging devices and endoscopes based on coherent Raman scattering are promising for label-free optical diagnosis. Toward the development of these small-scale systems, a challenge arises from the design and fabrication of achromatic and high-end miniature optical components for both pump and Stokes laser wavelengths. Here, we report a metasurface converting a low-cost plano-convex lens into a water-immersion, nearly diffraction-limited and achromatic lens. The metasurface comprising amorphous silicon nanopillars is designed in a way that all incident rays arrive at the focus with the same phase and group delay, leading to corrections of monochromatic and chromatic aberrations of the refractive lens, respectively. Compared to the case without the metasurface, the hybrid metasurface-refractive lens has higher Strehl ratios than the plano-convex lens and a tighter depth of focus. The hybrid metasurface-refractive lens is utilized in spectroscopic stimulated Raman scattering and coherent anti-Stokes Raman scattering imaging for the differentiation of two different polymer microbeads. Subsequently, the hybrid metalens is harnessed for volumetric coherent Raman scattering imaging of bead and tissue samples. Finally, we discuss possible approaches to integrate such hybrid metalens in a miniature scanning system for label-free coherent Raman scattering endoscopes.

Recent work on mid-infrared (MIR) detection through the process of non-degenerate two-photon absorption (NTA) in semiconducting materials has shown that wide-field MIR imaging can be achieved with standard Si cameras. While this approach enables MIR imaging at high pixel densities, the low nonlinear absorption coefficient of Si prevents fast NTA-based imaging at lower illumination doses. Here, we overcome this limitation by using InGaAs as the photosensor. Taking advantage of the much higher nonlinear absorption coefficient of this direct bandgap semiconductor, we demonstrate high-speed MIR imaging up to 500 fps with under 1 ms exposure per frame, enabling 2D or 3D mapping without pre- or post-processing of the image.

The microfluidic-based, label-free image-guided cell sorter offers a low-cost, high information content, and disposable solution that overcomes many limitations in conventional cell sorters. However, flow confinement for most microfluidic devices is generally only one-dimensional using sheath flow. As a result, the equilibrium distribution of cells spreads beyond the focal plane of commonly used Gaussian laser excitation beams, resulting in a large number of blurred images that hinder subsequent cell sorting based on cell image features. To address this issue, we present a Bessel-Gaussian beam image-guided cell sorter with an ultra-long depth of focus, enabling focused images of >85% of passing cells. This system features label-free sorting capabilities based on features extracted from the output temporal waveform of a photomultiplier tube (PMT) detector. For the sorting of polystyrene beads, SKNO1 leukemia cells, and green algae, our results indicate a sorting purity of 97%, 97%, and 98%, respectively, showing that the temporal waveforms from the PMT outputs have strong correlations with cell image features. These correlations are also confirmed by off-line reconstructed cell images from a temporal-spatial transformation algorithm tailored to the scanning Bessel-Gaussian beam.

Inadequate myelination in the central nervous system is associated with neurodevelopmental complications. Thus, quantitative, high spatial resolution measurements of myelin levels are highly desirable. We used spatial light interference microcopy (SLIM), a highly sensitive quantitative phase imaging (QPI) technique, to correlate the dry mass content of myelin in piglet brain tissue with dietary changes and gestational size. We combined SLIM micrographs with an artificial intelligence (AI) classifying model that allows us to discern subtle disparities in myelin distributions with high accuracy. This concept of combining QPI label-free data with AI for the purpose of extracting molecular specificity has recently been introduced by our laboratory as phase imaging with computational specificity. Training on 8000 SLIM images of piglet brain tissue with the 71-layer transfer learning model Xception, we created a two-parameter classification to differentiate gestational size and diet type with an accuracy of 82% and 80%, respectively. To our knowledge, this type of evaluation is impossible to perform by an expert pathologist or other techniques.

Nucleic acids and proteins are the two most important target types used in molecular diagnostics. In many instances, simultaneous sensitive and accurate detection of both biomarkers from the same sample would be desirable, but standard detection methods are highly optimized for one type and not cross-compatible. Here, we report the simultaneous multiplexed detection of SARS-CoV-2 RNAs and antigens with single molecule sensitivity. Both analytes are isolated and labeled using a single bead-based solid-phase extraction protocol, followed by fluorescence detection on a multi-channel optofluidic waveguide chip. Direct amplification-free detection of both biomarkers from nasopharyngeal swab samples is demonstrated with single molecule detection sensitivity, opening the door for ultrasensitive dual-target analysis in infectious disease diagnosis, oncology, and other applications.

Quantitative phase imaging (QPI) has been widely applied in characterizing cells and tissues. Spatial light interference microscopy (SLIM) is a highly sensitive QPI method due to its partially coherent illumination and common path interferometry geometry. However, SLIM's acquisition rate is limited because of the four-frame phase-shifting scheme. On the other hand, off-axis methods such as diffraction phase microscopy (DPM) allow for single-shot QPI. However, the laser-based DPM system is plagued by spatial noise due to speckles and multiple reflections. In a parallel development, deep learning was proven valuable in the field of bioimaging, especially due to its ability to translate one form of contrast into another. Here, we propose using deep learning to produce synthetic, SLIM-quality, and high-sensitivity phase maps from DPM using single-shot images as the input. We used an inverted microscope with its two ports connected to the DPM and SLIM modules such that we have access to the two types of images on the same field of view. We constructed a deep learning model based on U-net and trained on over 1000 pairs of DPM and SLIM images. The model learned to remove the speckles in laser DPM and overcame the background phase noise in both the test set and new data. The average peak signal-to-noise ratio, Pearson correlation coefficient, and structural similarity index measure were 29.97, 0.79, and 0.82 for the test dataset. Furthermore, we implemented the neural network inference into the live acquisition software, which now allows a DPM user to observe in real-time an extremely low-noise phase image. We demonstrated this principle of computational interference microscopy imaging using blood smears, as they contain both erythrocytes and leukocytes, under static and dynamic conditions.

Microresonator frequency combs and their design versatility have revolutionized research areas from data communication to exoplanet searches. While microcombs in the 1550 nm band are well documented, there is interest in using microcombs in other bands. Here, we demonstrate the formation and spectral control of normal-dispersion dark soliton microcombs at 1064 nm. We generate 200 GHz repetition rate microcombs by inducing a photonic bandgap of the microresonator mode for the pump laser with a photonic crystal. We perform the experiments with normal-dispersion microresonators made from TaO and explore unique soliton pulse shapes and operating behaviors. By adjusting the resonator dispersion through its nanostructured geometry, we demonstrate control over the spectral bandwidth of these combs, and we employ numerical modeling to understand their existence range. Our results highlight how photonic design enables microcomb spectra tailoring across wide wavelength ranges, offering potential in bioimaging, spectroscopy, and photonic-atomic quantum technologies.